GSM7474450: Mid thoracic spinal cord, drug_gfp_9 (rnaseq); Mus musculus; RNA-Seq
For snRNA-seq experiment, single-nucleus dissociation of the mouse lumbar spinal cord was performed according to our established procedures. Following euthanasia by isoflurane inhalation and cervical dislocation, the spinal cord injury site was immediately dissected and frozen on dry ice. Spinal cords were doused in 500 µL sucrose buffer (0.32 M sucrose, 10 mM HEPES [pH 8.0], 5 mM CaCl2, 3 mM Mg acetate, 0.1 mM EDTA, 1 mM DTT) and 0.1% Triton X-100 with the Kontes Dounce Tissue Grinder. 2 mL of sucrose buffer was then added and filtered through a 40 µm cell strainer. The lysate was centrifuged at 3200 g for 10 min at 4°C. The supernatant was then decanted, and 3 mL of sucrose buffer was added to the pellet for 1 min. We homogenized the pellet using an Ultra-Turrax and 12.5 mL of density buffer (1 M sucrose, 10 mM HEPES [pH 8.0], 3 mM Mg acetate, 1 mM DTT) was added below the nuclei layer. The tube was centrifuged at 3200 g at 4°C and supernatant poured off. Nuclei on the bottom half of the tube wall were collected with 100 µL PBS with 0.04% BSA and 0.2 U/µL RNase inhibitor. Finally, nuclei were resuspended through a 30 µm strainer, and adjusted to 1000 nuclei/µL. snRNA-seq library preparation was carried out using the 10x Genomics Chromium Multiome ATAC + Gene Expression Kit Version 3. The nuclei suspension was added to the Chromium RT mix to achieve loading numbers of 10,000 nuclei. For downstream cDNA synthesis (13 PCR cycles), library preparation and sequencing, the manufacturer's instructions were followed. For spatial transcriptomics, we carried out two separate experiments to study the cytoarchitecture of the lesion microenvironment after SCI. First, we prepared sections from uninjured mice, 7 days and 2 months after crush SCI. For each experimental condition, we prepared sections from the lesion epicenter of three independent biological replicates. Second, to prepare our four-dimensional spatiotemporal atlas, we collected sections throughout the entire spinal cord of mice from each of the three experimental conditions. The spinal cord injury sites of mice were embedded in OCT and cryosections were generated at 10 µm at −20 °C. For the four-dimensional atlas, every fifth section was collected throughout the entire dorsoventral axis of each spinal cord. Sections were immediately placed on chilled Visium Tissue Optimization Slides or Visium Spatial Gene Expression Slides. We used 10x Genomics Chromium Single Cell Gene Expression Kit Version 3.1 kit to profile the snRNA sequencing and Visium Spatial Gene Expression to profile the spatial transcriptomics.